胃癌患者胃液中特異性熒光物質(zhì)初步確定

1、胃癌患者胃液中特異性熒光物質(zhì)初步確定,研究背景,1990年, 我中心開(kāi)始致力于通過(guò)檢測(cè)血清和胃液的熒光光譜來(lái)診斷胃癌的系列研究。先后利用288 nm 紫外光作為激發(fā)光, 對(duì)500例各種胃內(nèi)疾患患者的胃液進(jìn)行高效液相熒光光譜檢測(cè), 發(fā)現(xiàn)胃癌患者胃液中存在兩種特異性熒光物質(zhì), 其含量較胃內(nèi)良性病變明顯增多。,研究背景,在上述實(shí)驗(yàn)的基礎(chǔ)上, 我們?cè)噲D通過(guò)聯(lián)合使用高效液相及質(zhì)譜技術(shù)分析高效液相熒光光譜中保留時(shí)間為11 .5 ～12 .5 min

2、 的熒光物質(zhì)成分, 從而對(duì)良惡性胃病患者胃液高效液相熒光光譜差異的物質(zhì)進(jìn)行初步分析。,研究目的,從胃癌患者胃液中發(fā)現(xiàn)特異性熒光物質(zhì) ,并以此來(lái)獲得腫瘤標(biāo)志物。,研究方法,收集 10例胃癌患者與 10例非胃癌患者的胃液 ,濃縮后行高效液相分析 , 并收集高效液相熒光光譜中保留時(shí)間為 11.5～ 12 .5min的熒光物質(zhì) ; 再經(jīng)質(zhì)譜鑒定 , 所得結(jié)果與L-色氨酸的質(zhì)譜比較 , 最終確定這種熒光物質(zhì)的主要組分。,研究結(jié)果,在高效液相

3、的熒光光譜保留時(shí)間為 11.5～ 12 .5min時(shí) , 胃癌患者胃液中有特異性熒光物質(zhì) ,其含量較其他胃內(nèi)良性病變患者明顯增多。,胃良、惡性病變患者濃縮胃液的高效液相熒光光譜,,,,研究結(jié)果,空白對(duì)照的一級(jí)質(zhì)譜圖中有共同質(zhì)荷比為279及288 的峰;10 例胃癌患者的一級(jí)質(zhì)譜圖中, 均出現(xiàn)質(zhì)荷比為205 , 217 , 279 , 288 , 404 , 790 及811 的峰, 有些還出現(xiàn)409。而10 例胃內(nèi)良性病變患者的一

4、級(jí)質(zhì)譜圖中, 均出現(xiàn)質(zhì)荷比為279 , 404 ,790 及811 的峰。,胃良、惡性病變患者胃液熒光峰收集樣品的一級(jí)質(zhì)譜圖,217,205,,,研究結(jié)果,推測(cè)胃癌患者胃液中含量較多的特殊熒光物質(zhì)可能對(duì)應(yīng)的是質(zhì)荷比為205 及217 的峰。經(jīng)質(zhì)譜鑒定后顯示 ,胃癌患者胃液中質(zhì)荷比為 205的物質(zhì)與L-色氨酸的三級(jí)質(zhì)譜完全一致 , 證實(shí)該物質(zhì)為L(zhǎng)-色氨酸（L-tryptophan）。,討論,近年來(lái)經(jīng)大量研究證實(shí), 機(jī)體內(nèi)存在許多內(nèi)源性

5、熒光物質(zhì), 它們形成的綜合熒光光譜, 在良性病變及腫瘤患者之間確實(shí)存在差異。而體液是腫瘤發(fā)生、演進(jìn)及轉(zhuǎn)移的內(nèi)環(huán)境, 腫瘤生長(zhǎng)過(guò)程中分泌及代謝產(chǎn)物會(huì)直接影響體液的組成。對(duì)體液進(jìn)行熒光檢測(cè), 有可能發(fā)現(xiàn)良惡性之間的差異。,討論,在本實(shí)驗(yàn)條件下, 對(duì)應(yīng)著高效液相保留時(shí)間為3 .5 ～ 4 .5 min 及11 .5 ～ 12 .5 min 的兩種物質(zhì), 利用高效液相技術(shù), 可以將胃液中保留時(shí)間在11 .5 ～ 12 .5 min 的特異性

6、熒光成分分離出來(lái), 減少其他物質(zhì)的干擾, 并可進(jìn)一步行質(zhì)譜分析。,討論,對(duì)比實(shí)驗(yàn)中, 從胃癌及胃內(nèi)良性病變患者的質(zhì)譜圖發(fā)現(xiàn), 兩者均為混合物。證實(shí)在液相保留時(shí)間為11 .5 ～ 12 .5 min 的收集物中, 同時(shí)存在熒光及無(wú)熒光的物質(zhì)。而胃癌患者獨(dú)有質(zhì)荷比為205 及217的峰。據(jù)此推測(cè), 胃癌患者特異性熒光物質(zhì)的相對(duì)分子質(zhì)量可能為204 或216 。,討論,L -色氨酸是一種重要的可產(chǎn)生熒光的芳香氨基酸。其相對(duì)分子質(zhì)量為204

7、 .2 , 本實(shí)驗(yàn)證實(shí), 它與胃癌特異性熒光物質(zhì)之一的保留時(shí)間一致。與質(zhì)荷比為205 物質(zhì)的一、二及三級(jí)質(zhì)譜的圖形完全一致。可以肯定, L-色氨酸是胃癌患者胃液中特異性熒光物質(zhì)的組成成分。,討論,質(zhì)荷比為217 的物質(zhì)也有可能參與熒光峰的形成。且其二級(jí)及三級(jí)質(zhì)譜與L-色氨酸一致。故懷疑它為一種與L-色氨酸相類(lèi)似的物質(zhì), 其實(shí)際相對(duì)分子質(zhì)量為216 。但因缺乏與本質(zhì)譜配套的解析標(biāo)準(zhǔn)譜庫(kù),目前無(wú)法進(jìn)一步搞清其結(jié)構(gòu)。,討論,對(duì)質(zhì)譜圖

8、中其他峰代表的物質(zhì), 也還應(yīng)明確其結(jié)構(gòu)及熒光特性。對(duì)高效液相保留時(shí)間在3 .5 ～ 4 .5 min 的物質(zhì), 尚有待進(jìn)一步確認(rèn)。,結(jié)論,本實(shí)驗(yàn)嘗試聯(lián)合使用高效液相和質(zhì)譜技術(shù)來(lái)分析胃液中一種腫瘤標(biāo)志物的方法。對(duì)新的胃癌腫瘤標(biāo)志物的發(fā)現(xiàn)起鋪墊作用, 具有廣泛的應(yīng)用前景。對(duì)揭示腫瘤發(fā)生、演進(jìn)及代謝的生化過(guò)程有可能起關(guān)鍵作用。,李淵,林三仁,周麗雅,彭嘉柔,郭慧蘭. 胃癌患者胃液中特異性熒光物質(zhì)的初步確定[J]. 中華消化雜志,2003

9、,01:11-14.,,,,,Three aromatic amino acids in gastric juice as potential biomarkers for gastric malignancies,BACKGROUND,For screening early-stage gastric malignancies, the existing

10、serum biomarkers have limited sensitivity and specificity. Gastric juice biomarkers are scarce and require further investigation.,研究背景,對(duì)于篩查早期胃惡性腫瘤，現(xiàn)存的血清學(xué)生物標(biāo)志物在敏感性和特異性方面都有其局限性。胃液生物標(biāo)志物稀缺，需要進(jìn)一步調(diào)查。,OBJECTIVE,We d

11、ivided this study on searching potential biomarkers into four parts: (1) detection of differential spectroscopy and HPLC, (2) identification and validation of differential peaks using LC/MS and NMR, (3) quantification of

12、 potential biomarkers, and (4) establishment of diagnostic detection.,研究目的,我們將這個(gè)篩選潛在的生物標(biāo)記物的研究分為四個(gè)部分：（1）聯(lián)合使用熒光光譜和高效液相色譜法檢測(cè)良惡性胃病患者胃液熒光光譜；（2）使用液相/質(zhì)譜和核磁共振的方法來(lái)識(shí)別和驗(yàn)證良惡性胃病患者胃液熒光光譜的區(qū)別；（3）定量研究潛在的生物標(biāo)記物；（4）建立診斷檢測(cè)。,METHODS,In this s

13、tudy, discriminating fluorescence biomarkers were isolated and identified from gastric juice by using high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS), and validated using

14、 nuclear magnetic resonance (NMR).,研究方法,本研究中應(yīng)用高效液相 (HPLC) ，液相/質(zhì)譜（LC/MS）的方法，分離并識(shí)別差異的胃液熒光標(biāo)志物，并且用核磁共振（NMR）的方法進(jìn)行了驗(yàn)證。,RESULTS,The fluorescence spectroscopy of diluted gastric juice indicates that the FI of P1 from patients wi

15、th gastric malignancies was significantly different from that in patients with benign gastric lesions (Fig. 1a and b).,研究結(jié)果,稀釋胃液固有熒光光譜表明，胃惡性腫瘤患者FIP1與胃良性病變者明顯不同（圖1A，B）。,Fig. 1. Discovery of fluorescence biomarkers. 圖1 熒

16、光標(biāo)志物的發(fā)現(xiàn)Fluorescence emission spectra (excitation at 288 nm) of diluted gastric juice from the benign (a) and malignant (b) groups.稀釋胃液的熒光激發(fā)光譜(激發(fā)光波長(zhǎng)為288納米)，良性(a)和惡性(b)組。,,,,,RESULTS,HPLC was used for the discovery of va

17、luable biomarkers in the gastric juice. The chromatograms are shown in Fig. 1c–f. By comparing the chromatograms for the benign and malignant groups, the discriminating biomarkers could be associated with peaks I, II,

18、III and IV, which were eluted around 8, 12, 16.5 and 33 min, respectively.,研究結(jié)果,采用高效液相色譜法測(cè)定胃液中有價(jià)值的生物標(biāo)志物。色譜圖如圖1c–f所示。對(duì)良性組和惡性組色譜圖進(jìn)行比較，有助于鑒別I，II，III和IV相關(guān)峰的生物標(biāo)志物，它們的洗脫時(shí)間分別約為8，12，16.5和33分鐘。,Fig. 1. Discovery of fluorescence

19、biomarkers. 圖1 熒光標(biāo)志物的發(fā)現(xiàn)Fluorescence chromatograms (λ ex = 288 nm, λ em = 360 nm) of gastric juice from benign (c) and malignant (d) groups. 稀釋胃液的熒光質(zhì)譜圖(激發(fā)光波長(zhǎng)為288納米，發(fā)射光波長(zhǎng)為360納米)，良性(c)和惡性(d)組。,,,,,Fig. 1. Discovery of f

20、luorescence biomarkers. 圖1 熒光標(biāo)志物的發(fā)現(xiàn)UV chromatograms (λ = 205 nm) of gastric juice from benign (e) and malignant (f) groups.稀釋胃液的紫外質(zhì)譜圖(激發(fā)光波長(zhǎng)為205納米)，良性(e)和惡性(f)組。,,,,,,,,,,,,,,RESULTS,The fluorescence intensity (FI), t

21、yrosine, phenylalanine, tryptophan and total protein content were significantly higher in the gastric juice of patients with gastric malignancies (all P<0.01).,研究結(jié)果,胃惡性腫瘤患者胃液中酪氨酸、苯丙氨酸、色氨酸的熒光強(qiáng)度(FI)和血清總蛋白

22、含量明顯高于胃良性病變患者 (P值均<0.01)。,Fig. 2. (a) Scatter plot indicates values of tyrosine, phenylalanine, tryptophan, total protein and FI in gastric juice from benign and malignant groups. 圖2（a）散點(diǎn)圖顯示良性和惡性病變組患者胃液中酪氨酸、苯丙氨酸、色氨酸、

23、總蛋白和熒光強(qiáng)度（FI）值。,,,,RESULTS,With all P<0.001, the areas under the receiver operating characteristic curves of the biomarkers were tyrosine, 0.838; phenylalanine, 0.856; and tryptophan, 0.816. At a specificity of 79.4%,

24、the sensitivity for gastric malignancy detection with phenylalanine was 87.9% only.,研究結(jié)果,在所有P < 0.001的生物標(biāo)志物中,受試者工作特征曲線（ROC）下面積分別是: 酪氨酸,0.838;苯丙氨酸,0.856;色氨酸,0.816。在特異性為79.4%時(shí)，苯丙氨酸用于胃惡性腫瘤檢測(cè)的靈敏度為87.9%。,Fig. 2.

25、 (b) ROC curves for detection of gastric malignancies were plotted with the use of tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp), protein and FI. 圖2（b）使用酪氨酸(Tyr),苯丙氨酸(Phe),色氨酸(Trp),總蛋白和熒光強(qiáng)度（FI）繪制的受試者工作特征（ROC）曲線用

26、于檢測(cè)胃惡性腫瘤。,,,DISCUSSIONS,The elevation of aromatic amino acids in gastric juice may be explained as follows.First, malignant cells are extremely invasive, and they can synthesize and secrete many enzymes (e.g., matrix me

27、talloproteinases, MMP and proteinases) that could degrade the basement membrane and extracellular matrix.,討論,胃液芳香族氨基酸升高的機(jī)制如下：首先，惡性細(xì)胞的侵襲性極強(qiáng)，它們可以合成并分泌許多酶類(lèi)（例如，金屬基質(zhì)蛋白酶 MMP和蛋白酶）降解基底膜和細(xì)胞外基質(zhì)。,DISCUSSIONS,Collagen fibers and el

28、astin contain endogenous fluorophores (aromatic amino acid residues), which have fluorescence properties, and are rich in the basement membrane and the extracellular matrix.Therefore, the elevation of aromatic amino aci

29、ds in gastric juice might be a phenomenon that results from degradation of basement membrane and extracellular matrix.,討論,膠質(zhì)纖維和彈性蛋白包括內(nèi)源性熒光物質(zhì)（氨基酸殘留物），在細(xì)胞基底膜和細(xì)胞外基質(zhì)的含量豐富。因此，胃液氨基酸含量的升高可能預(yù)示著基底膜和細(xì)胞外基質(zhì)的降解。,DISCUSSIONS,Second,

30、due to increased protein synthesis and rapid growth, more essential amino acids (especially for aromatic amino acids) may be up-regulated in malignant tissue.Some cells show exfoliation or necrosis and release cytoplasm

31、 enriched with essential amino acids, which could enhance the aromatic amino acids levels in gastric juices.,討論,第二，因?yàn)閻鹤兘M織蛋白質(zhì)合成增加，生長(zhǎng)加快，所以需要上調(diào)必須氨基酸（尤其是芳香族氨基酸）的表達(dá)。一些脫落或壞死的細(xì)胞釋放必需氨基酸含量豐富的細(xì)胞基質(zhì)，可以提高胃液芳香族氨基酸的水平。,DISCUSSIONS,Thi

32、rd, excess reactive oxygen species (ROS) produced during chronic immune activation around tumor tissue may interrupt the conversion of phenylalanine to tyrosine. Hence, the phenylalanine concentration increases.Moreover

33、, amino-acid-metabolizing enzymes (e.g., indoleamine 2,3-dioxygenase and monoamine oxidase) that are expressed aberrantly in malignant cells, may affect the aromatic amino acids levels in gastric juice.,討論,第三，在腫瘤組織周?chē)l(fā)生的慢

34、性炎性反應(yīng)過(guò)程中產(chǎn)生的過(guò)多的活性氧簇（ROS）阻止了苯丙氨酸轉(zhuǎn)化為酪氨酸。因此，苯丙氨酸的濃度升高。此外，氨基酸代謝酶（例如，吲哚胺2,3-雙加氧酶和單胺氧化酶）在惡性組織細(xì)胞中表達(dá)異常，可能會(huì)影響胃液芳香族氨基酸的水平。,DISCUSSIONS,All the above causes could elevate the concentration of aromatic amino acids in the gastric jui

35、ce of patients with gastric malignancies.,討論,所有上述原因均可以提高胃惡性腫瘤患者胃液中芳香族氨基酸的含量。,CONCLUSIONS,Aromatic amino acids in gastric juices could be used as potential diagnostic biomarkers to screen gastric 

36、malignancies. It is a less-invasive and economical method compared to gastric biopsy.,結(jié)論,胃液芳香族氨基酸可以作為潛在的診斷生物標(biāo)記物用于篩查胃惡性腫瘤。這是一個(gè)比胃粘膜活檢侵襲性更小和更經(jīng)濟(jì)的方法。,Deng K, Lin S, Zhou L, et al. Three aromatic amino acids in gas